Colorimetric dry reagent test strips, also called “dipsticks” or “dip-and-read reagent strips” are widely used for analyses, particularly outside a laboratory, where there is a necessity for a fast and simple detection.
Test strips for detection of hydrogen peroxide appeared on the market decades ago as a secondary (standby) product of sophisticated multi-enzymatic dry reagent test strips used in clinical chemistry for analyses of pathologically significant substances (enzymatic substrates) in body fluids, such as the analysis of glucose, uric acid, cholesterol etc. The principle underlying these test strips is that a corresponding oxidase enzyme converts a substrate into a particular reaction product while releasing hydrogen peroxide, which in following steps is the subject of a colorimetric detection to confirm the presence of the reaction product. Subsequently the market recognized that similar chemistry could be used for the sensing and determination of hydrogen peroxide, per se.
Broadly, these test strips comprise a plastic strip, at one end of which is mounted an absorbent portion impregnated with the appropriate reactant mixture. The test strips are used by dipping the reactant-bearing end into the test sample, removing it and evaluating the color formed in the paper portion.
Typically, the absorbent portion of such test strip includes an bibulous absorbent material, such as a cellulosic material, carrying a reactant composition capable of the color reaction with the compound the interest, namely hydrogen peroxide, generated from the analyzed sample. Such reactant compositions comprise an enzyme peroxidase or peroxidase-like substance and a chromogen compound capable of forming color or changing color in the presence of hydrogen peroxide. When this reactant system contained in the test strip comes into the contact with hydrogen peroxide, the peroxidase catalyzes a reaction between the hydrogen peroxide and the color changing compound to produce an oxidized (colored) form of the compound. The visually detectable change of color indicates a positive result.
Without catalysis the oxidation of a chromogen by hydrogen peroxide is not possible or is so weak that analytical use is not possible. It has been estimated that the presence of a peroxidase or peroxidase-like compound as a catalyst increases the sensitivity of detection over a thousandfold. For that reason the catalytic agent plays crucial role in detection of hydrogen peroxide, especially in low concentrations.
Substances having peroxidase activity include the vegetable peroxidases, such as horseradish, turnip, soybean or potato peroxidase; further inorganic compounds such as iodide and molybdate salts, iron thiocyanate, iron tannate etc. Red blood cells, urohemin and other porphyrin substances also have peroxidative activity, as do other compounds or combinations of compounds (see e.g. U.S. Pat. Nos. 3,298,789; 2,981,606; and 4,361,648). Some inorganic salts with peroxidase-like activity provide, in comparison with enzyme peroxidase, a much lower activity, resulting in significantly lower sensitivity of the analysis, especially in methods where a quick result is important. Since test strips, in general, are intended to offer a fast analysis, in the order of seconds, the use of a peroxidase enzyme for catalysis is crucial and thus are preferred in tests where high sensitivity and fast result are requested.
Enzyme activity and stability are generally strictly limited by pH. The most suitable peroxidases for analytical devices are horseradish peroxidases; their optimal pH range is about 6.0-6.5 pH units and their pH stability range is between 5.0 to 10.0 pH units. Other peroxidase compositions are set forth, for example, in U.S. Pat. Nos. 2,981,606; 3,012,976; 3,335,069; 3,558,435; 3,627,698; 4,361,648; 4,427,770; EP 0 217 246; EP 0 268 167.
The peroxidase catalyzes the reaction between hydrogen peroxide and the chromogen/color changing compound to produce the oxidized form of the compound. The color changing compound can be any compound capable of producing a color in the contact with hydrogen peroxide and enzyme peroxidase or generally a substance having peroxidase activity, and can be divided into two basic groups, leuco dye type chromogens, which are oxidized to form color products; and coupling type chromogens, which are oxidized to colorless compounds and coupled with a suitable coupler to form a final color product. A reactant composition incorporating a chromogen can preferably also contain non-reactive supportive compounds, such as buffer, polymers and wetting agents to improve the test strip's properties.
Colorimetric test strips provide a suitable analytical tool, especially in so-called field analyses, where there is a need for simple, fast and sensitive tests. Test strips contain all the reagents necessary for the test on single test pad in dry form, which also provides an advantage in the transportation of the strips and makes testing fast and easy, even for people not skilled in analytical techniques. Moreover no attendant apparatus is required. Thus, peroxide test strips can be a suitable analytical tool for the field detection of organic peroxide explosives, such as triaceton triperoxide (TATP) and hexamethylene triperoxide diamine (HMTD), often used in home-made explosives in terrorists attacks. The obstacle against use of commercially available peroxide test strips for that purpose is the use of strong mineral acids for the needed degradation of a peroxide-based explosive to release hydrogen peroxide. Such an acid degradation reaction, also called an acid treatment, is a broadly used procedure for releasing the detectable analyte (hydrogen peroxide) from rigid organic molecules in various testing methods, such as fluorescent, chemiluminescent, electrochemical and spectrophotometric detections (see, e.g. Anal. Bioanal. Chem. (2011) 400:313-320; Inorg. Chem. (2008) 47:9748-50; Analyst (2010) 135:2085-91; and Talanta (2015) 143:191-197).
Strong mineral acids, such as sulfuric and hydrochloric acid, are most often used for the needed acid degradation of peroxide based organic molecules; e.g. HCl in concentrations of 6N up to 32%. Such acid treatments result in a pH of the mixture in the range 0-1 pH units. The dipping of a test strip directly into such an acidic sample mixture would destroy the enzyme immediately, resulting in complete loss of its catalytic activity. Since a peroxidase enzyme is readily and irreversibly deactivated under strong acidic conditions, an additional procedure step is always required to neutralize the acidic mixture and adjust its pH to a value where the enzyme would be able to catalyze the needed interaction between hydrogen peroxide and chromogen, which is in the range of 5-9 pH units.
Such procedures are currently performed in laboratory methods and analytical kits (e.g. as set forth in U.S. Pat. No. 6,767,717) which require neutralization before a peroxidase can be introduced into the test mixture, but for dry-reagent test strip application, where the priority is for a fast and simple field test, (literally a “pocket-test”), which can be used by non-experts, the need for such additional neutralization step is a serious drawback.